Project Summary Regulatory T cell (Treg) plays a critical role in maintaining immune system homeostasis and preventing autoimmunity and immunopathology. Compromised Treg function is linked to multiple autoimmune diseases including type 1 diabetes and multiple sclerosis. On the flip side, enrichment of Treg cells within tumors is thought to be a barrier to effective anti-tumor immune response. The development and maintenance of Treg cell lineage are dependent on transcription factor Foxp3, as loss of function mutations lead to severe lymphoproliferative disease in mice and humans. Thus, understanding the mechanisms that govern Foxp3 induction and stability may lead to the development of novel therapies for autoimmune disease and cancer. Previously, Dr. Zheng and colleagues identified three evolutionarily conserved cis-regulatory elements at the Foxp3 locus, which play unique roles in Treg differentiation or maintenance. To expand this line of research, Dr. Zheng?s group will use a CRISPR/Cas9 based approach named CREST-seq (cis-regulatory element scan by tiling-deletion and sequencing) to perform unbiased screen and functional assessment for distal cis-elements near the Foxp3 locus. A preliminary study showed there are potentially up to 50 cis-regulatory elements involved in the control of Foxp3 expression. In-depth characterization of these new cis-elements will advance the understandings on the regulatory circuitry of Foxp3 expression. Numerous studies generated annotations on millions of candidate regulatory elements for thousands of genes in Tregs, but understanding is incomplete on how these elements control their target genes that are kilo-bases or even mega-bases away. Preliminary studies on genome-wide chromosome looping in Tregs generated a collection of distal DNA loops that can potentially regulate Treg signature genes. Furthermore, these data suggest a novel role for Foxp3 in facilitating distal DNA looping. To better understand how cis-regulatory circuitry control Treg differentiation and function, Dr. Zheng?s group will identify and characterize the cis-elements that control Foxp3 expression by a tiling deletion based genetic screen (Aim 1) and explore how distal enhancers regulate gene expression in regulatory T cell through chromosome looping (Aim 2). Furthermore, they will investigate if Foxp3 facilitates chromosome looping to establish and maintain the Treg lineage. Since Treg cell is directly related to autoimmune diseases and cancer, Dr. Zheng?s study will provide insight into the molecular mechanisms of Treg development and function and offer new opportunities to manipulate Tregs for therapeutic purposes.